Nontypeable Haemophilus influenzae (NTHi) is an important cause of otitis media in children and of pneumonitis in adults with depressed resistance. There is no vaccine available for NTHi. Lipooligosaccharide (LOS) is a major surface antigen of NTHi and elicits bactericidal and opsonic antibodies. We prepared detoxified LOS (dLOS) protein conjugates from NTHi for use as an experimental vaccine. LOS from strain 9274 was treated with anhydrous hydrazine and its toxicity was reduced to clinically acceptable levels. dLOS was bound to tetanus toxoid or high molecular weight proteins from NTHi through a linker to form conjugates. The antigenicity of the conjugates was similar to that of the LOS alone as determined by double immunodiffusion or ELISA. Subcutaneous or intramuscular injection of the conjugates elicited a 28 to 486-fold rise of IgG antibodies in mice to the homologous LOS after 2 to 3 injections, and a 169 to 243-fold rise in rabbits after 2 injections. The immunogenicity of the conjugates in mice and rabbits was enhanced by formulation with monophosphoryl lipid A plus trehalose dimycolate. In rabbits, conjugate-induced LOS antibodies induced complement-mediated bactericidal activity against the homologous strain 9274 and the heterologous strain 3189. These results indicate that a detoxified LOS-protein conjugate is a candidate vaccine for otitis media and pneumonitis caused by NTHi.